The present invention is directed to methods of reducing tumor progression and/or metastasis. Matriptase and MTSP1 are serine proteases reported to be expressed in high levels in certain cancer cell lines.
According to one aspect, the present invention provides compounds which bind to the serine protease domain and are active as selective inhibitors of the serine protease activity of matriptase or MTSP1. These inhibitors can be used to inhibit the serine protease activity of matriptase or MTSP1 and thereby reduce harmful effects of its over-expression. Another aspect of the present invention is directed to the use of compounds that bind to the serine protease domain and selectively inhibit the serine protease activity of matriptase or MTSP1; such compounds may be used in the prevention and treatment of cancerous conditions and which decrease the growth of cancerous tumors and retard metastasis.
Cancer is the second leading cause of death in the United States, after heart disease (Boring, C. C. et al., 1993, CA Cancer J. Clin. 43:7), and develops in one in three Americans. One of every four Americans dies of cancer. Cancer is characterized primarily by an increase in the number of abnormal, or neoplastic, cells derived from a given normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which spread via the blood or lymphatic system to regional lymph nodes and to distant sites. The latter progression to malignancy is referred to as metastasis.
Cancer can be viewed as a breakdown in the communication between tumor cells and their environment, including their normal neighboring cells. Signals, both growth-stimulatory and growth-inhibitory, are routinely exchanged between cells within a tissue. Normally, cells do not divide in the absence of stimulatory signals, and likewise, will cease dividing in the presence of inhibitory signals. In a cancerous, or neoplastic, state, a cell acquires the ability to xe2x80x9cOverridexe2x80x9d these signals and to proliferate under conditions in which normal cells would not grow.
Tumor cells must acquire a number of distinct aberrant traits to proliferate. Reflecting this requirement is the fact that the genomes of certain well-studied tumors carry several different independently altered genes, including activated oncogenes and inactivated tumor suppressor genes. Each of these genetic changes appears to be responsible for imparting some of the traits that, in aggregate, represent the full neoplastic phenotype (Land, H. et al. 1983, Science 222:771; Ruley, H. E., 1983, Nature 304:602; Hunter, T. 1991, cell 64:249).
In addition to unhindered cell proliferation, cells must acquire several traits for tumor progression to occur. For example, early on in tumor progression, cells must evade the host immune system. Further, as tumor mass increases, the tumor must acquire vasculature to supply nourishment and remove metabolic waste. Additionally, cells must acquire an ability to invade adjacent tissue, and, ultimately, cells often acquire the capacity to metastasize to distant sites.
A variety of biochemical factors have been associated with different phases of metastases. Cell surface receptors for collagen, glycoproteins such as laminin, or proteoglycans, facilitate tumor cell attachment, an important step in invasion and metastases. Attachment then triggers the release of degradative enzymes which facilitate the penetration of tumor cells through tissue barriers. Once the tumor cells have entered the target tissue, specific growth factors are required for further proliferation.
One of the major characteristics of cancer cells is their ability to invade surrounding normal tissues and metastasize to distant body sites. It is the metastatic nature of malignant tumors that presents a great challenge to clinicians in terms of treatment, since the tumor is no longer localized to one area.
Tumor invasion (or progression) is a complex series of events, in which tumor cells detach from the primary tumor, break down the normal tissue surrounding it, and migrate into a blood or lymphatic vessel to be carried to a distant site. The breaking down of normal tissue barriers is accomplished by the elaboration of specific enzymes that degrade the proteins of the extracellular matrix that make up basement membranes and stromal components of tissues.
A class of extracellular matrix degrading enzymes has been identified called the matrix metalloproteinases (MMP). Two of the matrix metalloproteinases have been implicated in tumor invasion. The type IV collagenase has been correlated with the metastatic potential of tumor cells. (Liotta, et al., Nature, 284:67-68 (March, 1980)). It has been reported that the production of the matrix metalloproteinase stromelysin was associated with malignant tumors with metastatic potential. (McDonnell and Matrisian, Smnrs. in Cancer Biology 1:107-115 (1990); McDonnell and Matrisian, Cancer and Metastasis Reviews 9:309-319 (1990).)
The capacity of cancer cells to metastasize and invade tissue is facilitated by degradation of the basement membrane. Several proteinase enzymes have been reported to facilitate the process of invasion of tumor cells. One family of enzymes, MMPs, has been implicated as enhancing degradation of the basement membrane to allow tumorous cells to invade tissues. MMPs have been reported to differ in molecular weight and antigenic properties. Previously, two major metalloproteinases having molecular weights of about 70 kDa and 92 kDa have been studied. Both of these MMPs have been reported to enhance ability of tumor cells to metastasize. Two natural inhibitors of these enzymes known as tissue inhibitors of metalloproteinase (TIMP) have been identified. The inactivated unclipped collagenases are generally secreted as a complex with TIMP. Enzymatic activity of the 72 kDa and 92 kDa proteins has been reported to depend on secreted ratios of collagenase/TIMP.
Matriptase is a trypsin-like serine protease which has been isolated and cloned from T-47D human breast cancer cells. Matriptase has been isolated from T-47D cell-conditioned medium. (Lin, et al., J. Biol. Chem. 274(26):18231-18236 (1999).) Upon analysis of the cDNA, it was determined that the protease had 683 amino acids and contained three main structural regions: a serine protease domain near the carboxyl-terminal region, four tandem low-density lipoprotein receptor domains, and two tandem complement subcomponents C1r and C1s. Matriptase was reported to be a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. It was named xe2x80x9cmatriptasexe2x80x9d because of the ability of the protease to degrade the extra-cellular matrix and its trypsin-like activity. (Lin, et al., J. Bio. Chem. 274:18231-18236 (1999).)
Matriptase is reported to be a protease having activity in degrading extracellular matrix that is localized on the cell surface. When isolated from breast cancer cells (or T-47D cell conditioned medium), matriptase has been reported to be primarily in an uncomplexed form. Matriptase has been isolated from human milk. When isolated from human milk, matriptase was reported to be in one of two complexed forms, 95 kDa (the predominant form) and 110 kDa; uncomplexed matriptase was not detected. (Liu, et al., J. Biol. Chem. 274(26):18237-18242 (1999).) It has been proposed that matriptase exists as an uncomplexed protease when in its active state. In breast milk, matriptase has been reported to exist in complex with a fragment of hepatocyte growth factor inhibitor-1 (HAI-1), a Kuntz-type serine protease inhibitor having activity against trypsin-like serine proteases.
Ecotin and Ecotin M84R/M85R have been reported to be macromolecular inhibitors of serine proteases of the chymotrypsin fold and have been reported to inhibit ductal branching, morphogenesis and differentiation of the explanted ductal prostate. PC-3 is a cell line derived from prostate cancer epithelial cells. Ecotin and M84R/M85R ecotin were found to decrease tumor size and metastasis in PC-3 implanted nude mice. Studies to identify additional serine proteases made by cancer cells were done using PC-3 cells. By using PCR techniques and degenerate oligonucleotide primers, five independent serine protease cDNAs were reported isolated from PC-3 mRNA. A serine protease termed xe2x80x9cMT-SP1xe2x80x9d was cloned, its cDNA characterized and reported to encode a mosaic, transmembrane protease. (Takeuchi, et al., PNAS (US) 96:11054-11061 (1999).)
It was subsequently reported that the reported matriptase sequence was included in the translated sequence for the cDNA of MT-SP1. The matriptase cDNA was reported to be a partial MT-SP1 cDNA and to lack 516 of the coding nucleotides. (Takeuchi, et al., J. Biol. Chem 275:26333-26342 (2000).) Since the reported matriptase cDNA sequence encoded a possible initiating methionine, it was proposed that alternative splicing could yield a protein lacking the N-terminal region of MT-SP1.
Both matriptase and MT-SP1 are reported to demonstrate trypsin-like protease activity. MT-SP1 has been reported to be a Type II transmembrane protein with an extracellular protease domain. Studies on matriptase have reported that a portion of enzyme molecules were localized on the surfaces of cells.
Additional studies have investigated the substrate specificity of MT-SP1. These experiments have reported that protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator (sc-uPA) are macromolecular substrates of MT-SP1. PAR2 is reported to function in inflammation, cytoprotection and/or cell adhesion, while sc-uPa is reported to function in tumor cell invasion and metastasis.
Elevated proteolytic activity has been implicated in neoplastic progression. The role(s) of proteolytic enzymes, including serine proteases, in neoplastic progression are under study. Proteases have been proposed to contribute to the degradation of excellular matrix and to tissue remodeling and, thus, may assist in cancer invasion and metastasis.
A number of extracellular proteases have been reported and expression of some of these proteases has been said to correlate with tumor progression. (Mignatti, P. and Rifkin, D. B., Physiol. Rev. 73:161-195 (1993).)
In one aspect, the present invention relates to compounds which bind to the serine protease domain of matriptase or MTSP1 and which are selective inhibitors of the serine protease activity matriptase or MTSP1 and to their use in inhibiting the serine protease activity matriptase or MTSPI. As noted above, the reported sequence for MT-SP1 includes the reported matriptase sequence. We have cloned the serine protease domain and have determined that matriptase and MTSP1 have a common serine protease domain. Inhibition of serine protease activity of matriptase or MTSP1 is useful in the prevention and treatment of cancerous conditions, including having activity in retarding tumor progression and metastasis.
Matriptase and MTSP1 both have been reported to be trypsin-like serine proteases involved in the degradation of the extracellular matrix (ECM). Our cloning studies indicate that matriptase and MTSP1 share a common serine protease domain. Degradation of the extracellular matrix (basement membrane and interstitial stroma) is an important aspect of metastasis and is required for metastatic cancer cells to migrate through anatomical barriers and to invade tissues. Thus, breakdown of the extracellular matrix is a primary factor in progression of cancerous conditions. In one aspect, the present invention relates to compounds and their use for the selective inhibition of serine protease activity of matriptase or MTSP1 in order to decrease the cancer-related breakdown of the extracellular matrix.
The present invention is also directed to administration of a selective inhibitor of serine protease activity of matriptase or MTSP1 to an area of an animal suspected of having metastic cancer cells in order to retard tumor progression. Also, provided are pharmaceutical compositions which comprise a selective inhibitor of serine protease activity matriptase or MTSP1 and a pharmaceutically acceptable carrier.
The present invention also provides methods for the selection of the compounds that will selectively modulate the serine protease activity of matriptase or MTSP1.
Thus, according to one aspect of the present invention, provided are methods of detecting a compound which inhibits serine protease activity of matriptase or MTSP1 which comprises contacting said compound with a peptide comprising a recombinant serine protease domain derived from matriptase or MTSP1 and a substrate and measuring substrate hydrolysis. Preferably the serine protease domain comprises SEQ. ID. NO. 2 or an amino acid sequence having a serine protease activity and at least about 80% sequence identity to SEQ. ID. NO. 2. More preferably, the serine protease domain comprises an amino acid sequence having at least about 90%, even more preferably about 95% or more, sequence identity to SEQ. ID. NO. 2.
Alternatively, the present invention is directed to a method of screening a compound having activity in inhibiting serine protease activity of matriptase or MTSP1 which comprises determining whether said compound inhibits serine protease activity of a peptide comprising a recombinant serine protease domain derived from matriptase or MTSP1. Preferably said peptide comprises SEQ. ID. NO. 2 or an amino acid sequence having serine protease activity and at least about 80%, more preferably at least about 90%, and even more preferably 95% or more, sequence identity to SEQ. ID. NO. 2.
Also provided is a method of screening a compound for activity in inhibiting serine protease activity of matriptase or MTSP1 which comprises determining whether said compound inhibits activity of a peptide comprising a recombinant serine protease domain comprising SEQ. ID. NO. 2 or an amino acid sequence having serine protease activity and at least about 80% sequence identity to SEQ. ID. NO. 2. Preferably the amino acid sequence has at least about 90% sequence identity and more preferably 95% or more sequence identity to SEQ. ID. NO. 2.
According to a further aspect, the present invention provides a recombinant serine protease domain derived from matriptase or MTSP1 which comprises SEQ. ID. NO. 2 or an amino acid sequence having serine protease activity and at least about 80% sequence identity to SEQ. ID. NO. 2. Also provided is a recombinant serine protease domain which comprises SEQ. ID. NO. 2 or an amino acid sequence having serine protease activity and at least about 80% sequence identity to SEQ. ID. NO. 2. More preferably, the serine protease domain comprises an amino acid sequence having at least about 90% and more preferably about 95% or more sequence identity to SEQ. ID. NO. 2. Especially preferred is a serine protease domain which comprises SEQ. ID. NO. 2.
xe2x80x9cMatriptasexe2x80x9d is a trypsin-like serine protease active in the development of cancerous conditions, such as tumors and metastasis of cancer. Matriptase is a matrix degrading protease that is reported to be localized on the cell surface. Matriptase is reported to have 683 amino acids and contains three main structural regions, a serine protease domain near the carboxyl-terminal region, four tandem low-density lipoprotein receptor domains, and two tandem complement subcomponents C1r and C1s.
Membrane-type serine protease 1 (xe2x80x9cMT-SP1xe2x80x9d or xe2x80x9cMTSP1xe2x80x9d) refers to a serine protease originally cloned from the PC-3 cell line. MT-SP1 is reported to be a Type II transmembrane protein with an extracellular protease domain. The amino acid sequence reported for matriptase has been reported to be included within the translated sequence for the cDNA of MT-SP1. The cDNA of MT-SP1 is reported to have, in addition to regions coding for the three main structural regions described above for matriptase, an additional 516 nucleotides that make up a signal/anchor domain.
A xe2x80x9ccancerous conditionxe2x80x9d is one in which the patient has a progressive human cancer, such as, leukemia, lymphomas, human melanomas, breast, gastrointestinal, such as esophageal, stomach, colon, bowel, colorectal and rectal cancers, prostate, bladder, testicular, ovarian, uterine, cervical, brain, lung, bronchial, larynx, pharynx, pancreatic, thyroid, bone, and various types of skin cancers.
A xe2x80x9ctumor,xe2x80x9d also known as a neoplasm, is an abnormal growth of tissue resulting from uncontrolled cell replication, often related to cancer.
xe2x80x9cMetastasisxe2x80x9d is the spread of cancer from an original location to a new location in the body.